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Label-Free, Multiplexed, Single-Molecule Analysis of Protein-DNA Complexes with Nanopores
Abstract
Protein interactions with specific DNA sequences are crucial in the control of gene expression and the regulation of replication. Single-molecule methods offer excellent capabilities to unravel the mechanism and kinetics of these interactions. Here we develop a nanopore approach where a target DNA sequence is contained in a hairpin followed by a ssDNA. This system allows DNA-protein complexes to be distinguished from bare DNA molecules as they are pulled through a single nanopore detector, providing both equilibrium and kinetic information. We show that this approach can be used to test the inhibitory effect of small molecules on complex formation, and their mechanisms of action. In a proof of concept, we use DNAs with different sequence patterns to probe the ability of the nanopore to distinguish the effects of an inhibitor in a complex mixture of target DNAs and proteins. We anticipate that the use of this technology with arrays of thousands of nanopores will contribute to the development of transcription factor binding inhibitors.
... By measuring dynamic changes of current through the nanopore, this technique has also demonstrated great potential to detect biomolecular structures. When a protein (51), DNA (30,52,53), RNA (54)(55)(56), or nucleic acid/protein complex (57,58) occludes the nanopore under a transmembrane voltage, their structures can characteristically modulate the ion current through the pore. The resulting nanopore current pattern or "signature" can be analyzed to discriminate the molecular structure (59)(60)(61)(62)(63)(64). ...
Nucleic acids can undergo conformational changes upon binding small molecules. These conformational changes can be exploited to develop new therapeutic strategies through control of gene expression or triggering of cellular responses and can also be used to develop sensors for small molecules such as neurotransmitters. Many analytical approaches can detect dynamic conformational change of nucleic acids, but they need labeling, are expensive, and have limited time resolution. The nanopore approach can provide a conformational snapshot for each nucleic acid molecule detected, but has not been reported to detect dynamic nucleic acid conformational change in response to small -molecule binding. Here we demonstrate a modular, label-free, nucleic acid-docked nanopore capable of revealing time-resolved, small molecule-induced, single nucleic acid molecule conformational transitions with millisecond resolution. By using the dopamine-, serotonin-, and theophylline-binding aptamers as testbeds, we found that these nucleic acids scaffolds can be noncovalently docked inside the MspA protein pore by a cluster of site-specific charged residues. This docking mechanism enables the ion current through the pore to characteristically vary as the aptamer undergoes conformational changes, resulting in a sequence of current fluctuations that report binding and release of single ligand molecules from the aptamer. This nanopore tool can quantify specific ligands such as neurotransmitters, elucidate nucleic acid-ligand interactions, and pinpoint the nucleic acid motifs for ligand binding, showing the potential for small molecule biosensing, drug discovery assayed via RNA and DNA conformational changes, and the design of artificial riboswitch effectors in synthetic biology.
... Biological nanopore instruments with representative ssDNA and protein-bound DNA events[67,93]. Image reprinted from[67] with permission of the publisher (CCC License ID: 600061571, 25 Nov 2021). ...
Nanopore sequencing to resolve problems associated with tumor heterogeneity.
... Biological nanopore instruments with representative ssDNA and protein-bound DNA events[67,93]. Image reprinted from[67] with permission of the publisher (CCC License ID: 600061571, 25 Nov 2021). ...
Nanopore sequencing has brought the technology to the next generation in the science of sequencing. This is achieved through research advancing on: pore efficiency, creating mechanisms to control DNA translocation, enhancing signal-to-noise ratio, and expanding to long-read ranges. Heterogeneity regarding epigenetics would be broad as mutations in the epigenome are sensitive to cause new challenges in cancer research. Epigenetic enzymes which catalyze DNA methylation and histone modification are dysregulated in cancer cells and cause numerous heterogeneous clones to evolve. Detection of this heterogeneity in these clones plays an indispensable role in the treatment of various cancer types. With single-cell profiling, the nanopore sequencing technology could provide a simple sequence at long reads and is expected to be used soon at the bedside or doctor’s office. Here, we review the advancements of nanopore sequencing and its use in the detection of epigenetic heterogeneity in cancer.
... Another area for potential improvement is classifier prediction accuracy, which can be influenced by classifier type and the number of training examples ( Supplementary Fig. 19) or by imposing a confidence threshold (Supplementary Fig. 13). Future work will be aimed at: 1) further expansion of the barcode space, for example, with chemical modifications to the DNA that could expand the dynamic range of barcode signal space 40 , and/or the ability to read sequential barcode regions within a single output strand 25,41,42 ; and 2) increasing the reaction speed and sensitivity of DSD reactions, for example by spatially-localizing DNA components to the nanopore sensor membrane 43,44 . Increasing the scale and speed of our detection strategy for more complex molecular computing architectures, such as cascaded circuits or oscillators, will further take advantage of our method's ability to generate both multiplexed and kinetic readouts. ...
DNA has emerged as a powerful substrate for programming information processing machines at the nanoscale. Among the DNA computing primitives used today, DNA strand displacement (DSD) is arguably the most popular, with DSD-based circuit applications ranging from disease diagnostics to molecular artificial neural networks. The outputs of DSD circuits are generally read using fluorescence spectroscopy. However, due to the spectral overlap of typical small-molecule fluorescent reporters, the number of unique outputs that can be detected in parallel is limited, requiring complex optical setups or spatial isolation of reactions to make output bandwidths scalable. Here, we present a multiplexable sequencing-free readout method that enables real-time, kinetic measurement of DSD circuit activity through highly parallel, direct detection of barcoded output strands using nanopore sensor array technology (Oxford Nanopore Technologies’ MinION device). These results increase DSD output bandwidth by an order of magnitude over what is currently feasible with fluorescence spectroscopy. Toe-hold-mediated strand displacement (DSD) is a widely used molecular tool in applications such as DNA computing and nucleic acid diagnostics. Here the authors characterize dozens of orthogonal barcode sequences that can be used for monitoring the output kinetics of multiplexed DSD reactions in real-time using a commercially-available portable nanopore array device.
... Finally, the remaining challenges for protein nanopore based SMS and the promising future research directions are summarized. Note that this review focuses specifically on the progress and prospects of single-molecule sensing by protein pores, many other exciting studies are therefore not included, such as the nanopore -1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 A c c e p t e d M a n u s c r i p t F o r R e v i e w O n l y enabled sequencing of DNA 68 or protein 69 , measurement of dynamic interaction 70,71 , and analysis of reaction mechanism 72,73 . In the meantime, for readers who are interested in the solid-state or other types of membrane nanopores, please check the recent reviews by Tim Albrecht 10 and Stefan Howorka 51 . ...
The recent development of single-molecule sensors (SMS), which detect individual targets one at a time, allows determination of ultra-low concentrations of structurally similar compounds from a complex matrix. Protein nanopores are one of the earliest methods able to resolve the signal from a single molecule, and have already been successfully employed in commercial DNA sequencers. The protein nanopore based SMS, however, remains challenging, largely because the quantitative single-molecule analysis requires recording a sufficient number of signals for statistical significance within a reasonable time frame, thus restricting the lower limit of detection. This review aims to critically evaluate the strategies developed in this field over the last two decades. The measurement principle of nanopore SMS is first elucidated, followed by a systematic examination of the eight common protein pores, and a comprehensive assessment of the major types of sensing applications. A particular emphasis is placed on the intrinsic relationship between the size and charge of protein nanopores and their sensing capabilities for different kinds of analytes. Innovative approaches to lift the performance of nanopore SMS are also analyzed in detail, with a prediction at the end of the most promising future applications.
... Celaya and Perales-Calvo developed a nanopore method to distinguish naked DNA from DNA-protein complexes [98], which tests the inhibitory effect of small molecules on complex formation and the mechanism of action and facilitates the development of transcription factor binding inhibitors. In 2019, Kaur et al. detected the binding RNA polymerase (RNAP) on 48.5 kbp (16.5 µm) λDNA using silicon nitride-based nanopore [99]. ...
Protein is an important component of all the cells and tissues of the human body and is the material basis of life. Its content, sequence, and spatial structure have a great impact on proteomics and human biology. It can reflect the important information of normal or pathophysiological processes and promote the development of new diagnoses and treatment methods. However, the current techniques of proteomics for protein analysis are limited by chemical modifications, large sample sizes, or cumbersome operations. Solving this problem requires overcoming huge challenges. Nanopore single molecule detection technology overcomes this shortcoming. As a new sensing technology, it has the advantages of no labeling, high sensitivity, fast detection speed, real-time monitoring, and simple operation. It is widely used in gene sequencing, detection of peptides and proteins, markers and microorganisms, and other biomolecules and metal ions. Therefore, based on the advantages of novel nanopore single-molecule detection technology, its application to protein sequence detection and structure recognition has also been proposed and developed. In this paper, the application of nanopore single-molecule detection technology in protein detection in recent years is reviewed, and its development prospect is investigated.
... Moreover, TMD TraJ CD TrwB showed negative dominance in the presence of the native T4CPs TrwB R388 or TraJ pKM101 . Since it has been reported that TMD TraJ can interact with both T4SS R388 and T4SS pKM101 (Llosa et al., 2003;De Paz et al., 2010;Celaya et al., 2017), a possible explanation for the observed negative dominance could be that competition for the T4SS occurred. According to this hypothesis, TMD TraJ CD TrwB would have interacted with the secretion channel, sequestering it from interacting with the native T4CPs and reducing the conjugative rate of the wild type system. ...
Type IV Coupling Proteins (T4CPs) are essential elements in many type IV secretion systems (T4SSs). The members of this family display sequence, length, and domain architecture heterogeneity, being the conserved Nucleotide-Binding Domain the motif that defines them. In addition, most T4CPs contain a Transmembrane Domain (TMD) in the amino end and an All-Alpha Domain facing the cytoplasm. Additionally, a few T4CPs present a variable domain at the carboxyl end. The structural paradigm of this family is TrwBR388, the T4CP of conjugative plasmid R388. This protein has been widely studied, in particular the role of the TMD on the different characteristics of TrwBR388. To gain knowledge about T4CPs and their TMD, in this work a chimeric protein containing the TMD of TraJpKM101 and the cytosolic domain of TrwBR388 has been constructed. Additionally, one of the few T4CPs of mobilizable plasmids, MobBCloDF13 of mobilizable plasmid CloDF13, together with its TMD-less mutant MobBΔTMD have been studied. Mating studies showed that the chimeric protein is functional in vivo and that it exerted negative dominance against the native proteins TrwBR388 and TraJpKM101. Also, it was observed that the TMD of MobBCloDF13 is essential for the mobilization of CloDF13 plasmid. Analysis of the secondary structure components showed that the presence of a heterologous TMD alters the structure of the cytosolic domain in the chimeric protein. On the contrary, the absence of the TMD in MobBCloDF13 does not affect the secondary structure of its cytosolic domain. Subcellular localization studies showed that T4CPs have a unipolar or bipolar location, which is enhanced by the presence of the remaining proteins of the conjugative system. Unlike what has been described for TrwBR388, the TMD is not an essential element for the polar location of MobBCloDF13. The main conclusion is that the characteristics described for the paradigmatic TrwBR388 T4CP should not be ascribed to the whole T4CP family. Specifically, it has been proven that the mobilizable plasmid-related MobBCloDF13 presents different characteristics regarding the role of its TMD. This work will contribute to better understand the T4CP family, a key element in bacterial conjugation, the main mechanism responsible for antibiotic resistance spread.
... Other translocation configurations can also be recognised, including monomer, dimer and clustered particles [238], folded/multi-folded DNA [239], knots on DNA [240], protein-DNA complex [241], DNA protrusion [242] and specially structured RNA/DNA [243]. These fingerprint features can be used to detect target analytes [244,245] or identify specific interactions [225,246,247]. Various schemes have been developed to utilise the DNA protrusions, dumbbell hairpins and bound proteins generated current notches as symbols to code, carry and store digitalised information [248][249][250][251]. ...
Solid-state nanopore (SSNP) technology presents an emerging single-molecule based analytical tool for separation and analysis of biomolecules or nanoparticles. Different prominent approaches have been pursued to attain the anticipated detection performance: process
innovation to achieve pore size matching the physical dimensions of biomolecules so as to boost signal; electrolyte management to control translocation speed so as to improve signal quality; surface functionalisation to amplify molecular differences so as to enhance specificity; and implementation of additional, complementary means, such as optical, to manipulate the translocation so as to increase data fidelity. This review focuses on the fundamentals pertaining to the physical processes involved in nanopore sensing based on SSNPs of distinct shapes. It also provides a comprehensive picture regarding challenges and development trends in putting nanopore-based molecular sensors in use. This effort is facilitated by establishing physical-phenomenological models supported by experiment and numerical simulation. To assist the readership, the discussion starts from relatively simple cases and then develops towards complex systems, i.e. from open-pore state to analyte translocation and from single pores to pore arrays. Key physical parameter threading through these aspects is effective transport length that is simple to perceive and easy to calculate.
... Simultaneous detection of multiple tumor markers brings new opportunities for improving the accuracy of early cancer detection compared to the single-marker assay. [43][44][45] Recently, various platforms using nanomaterials for the detection of multiple targets have been developed, [46][47][48][49][50] but relatively few molecular probes have been developed that detect multiple tumor markers simultaneously. 51 Herein, we report a dual site fluorescence probe, CNN, to which a 4-nitrobenzene moiety and trimethyl-locked quinone propionic acid (Q3PA) were introduced as reaction units, as well as a fluorescence quencher. ...
Identifying cancer at the cellular level during an early stage offers the hope of greatly improved outcomes for cancer patients. As potential cancer biomarkers, nitroreductase (NTR) and human quinine oxidoreductase 1 (hNQO1) are overexpressed in many type of cancer cells. Simultaneous detection of these two biomarkers would benefit diagnostic precision in related cancers without yielding false positive results. Herein, based on a dye generated in situ strategy, a dual-enzyme-responsive probe, CNN, was rationally designed and synthesized by installing p-nitrobenzene and trimethyl-locked quinone propionic acid groups, which are specific for NTR and hNQO1, respectively, into a single fluorophore. This probe is only activated in the presence of both NTR and hNQO1 and produces a large fluorescence response, enabling the detection of both endogenous NTR and hNQO1 activity in living cells. The imaging results indicate that the CNN probe differentiates cancer cells (HeLa, MDA-MB-231 and HepG2 cells) from normal liver HL-7702 cells owing to the existence of relatively high endogenous levels of both biomarkers in these cancer cells.
... In the present study, we focused on the distinguishability of different AA in α-Hemolysin (αHL), the most widely employed pore in nanopore sensing 2,[18][19][20]35,37,38 . To this aim, as a preliminary case study, we analyzed the differences of homopeptide chains inserted in the αHL. ...
Single molecule protein sequencing would represent a disruptive burst in proteomic research with important biomedical impacts. Due to their success in DNA sequencing, nanopore based devices have been recently proposed as possible tools for the sequencing of peptide chains. One of the open questions in nanopore protein sequencing concerns the ability of such devices to provide different signals for all the 20 standard amino acids. Here, using equilibrium all-atom molecular dynamics simulations, we estimated the pore clogging in α-Hemolysin nanopore associated to 20 different homopeptides, one for each standard amino acid. Our results show that pore clogging is affected by amino acid volume, hydrophobicity and net charge. The equilibrium estimations are also supported by non-equilibrium runs for calculating the current blockades for selected homopeptides. Finally, we discuss the possibility to modify the α-Hemolysin nanopore, cutting a portion of the barrel region close to the trans side, to reduce spurious signals and, hence, to enhance the sensitivity of the nanopore.
... Due to their mechanical robustness, tunable size, and integration potential, solid-state nanopores emerge as promising label-free sensors for detection of single molecules such as DNAs [1][2][3][4][5], RNAs [6][7][8], proteins [9][10][11], and DNA-protein complexes [12][13][14]. The ionic current blockades during the molecule translocation could provide rich information about the molecule properties [1,2,15]. ...
We investigate the current transport characteristics in the electrolyte-dielectric-electrolyte structure commonly used in the in situ controlled breakdown (CBD) fabrication of solid-state nanopores. It is found that the stochastic breakdown process could lead to fidelity issues of false positives (an incorrect indication of a true nanopore formation) and false negatives (inability to detect initial nanopore formation). Robust and deterministic detection of initial physical breakdown to alleviate false positives and false negatives is critical for precise nanopore size control. To this end, we report a high fidelity moving Z-score method based CBD fabrication of solid-state nanopore. We demonstrate 100% success rate of realizing the initial nanopore conductance of 3 ± 1 nS (corresponds to size of 1.7 ± 0.6 nm) regardless of the dielectric membrane characteristics. Our study also elucidates the Joule heating is the dominant mechanism for electric field-based nanopore enlargement. Single DNA molecule sensing using nanopores fabricated by this method was successfully demonstrated. We anticipate the moving Z-score based CBD method could enable broader access to the solid state nanopore-based single molecule analysis.
... The transport of ions, molecules and polymers across constrictions such as pores, membranes or varyingsection micro-nano-channels is crucial for several biological as well as synthetic systems. For example, in biological cells ion channels control the uptake of ions from the environment [1] whereas, in resistive pulse sensing techniques, the interactions of colloidal particles [2] or macromolecules [3][4][5][6][7] with the nano-or micro-pore are measured from the variation of the electric conductance of the pore induced by the presence of the particle. Moreover, electro-osmotic flux can play a crucial role in molecule capturing in nanopores [8][9][10]. ...
We characterize the dynamics of an electrolyte embedded in a varying-section channel under the action of a constant external electrostatic field. By means of molecular dynamics simulations we determine the stationary density, charge and velocity profiles of the electrolyte. Our results show that when the Debye length is comparable to the width of the channel bottlenecks a concentration polarization along with two eddies sets inside the channel. Interestingly, upon increasing the external field, local electroneutrality breaks down and charge polarization sets leading to the onset of net dipolar field. This novel scenario, that cannot be captured by the standard approaches based on local electroneutrality, opens the route for the realization of novel micro and nano-fluidic devices.
... Utilizing the ionic current blockage caused by biomolecule translocation through nanopores, we can obtain information about the structure and dynamics of biomolecules [2]; this method has advantages such as high resolution, low cost, minimal sample damage, sequential direct reading, and excellent environmental compatibility and does not always require sample pretreatment. Nanopores are widely used in the detection of proteins [3][4][5][6][7], DNA-protein complexes [8,9], RNA [10], polymers [11,12], metal ions [13] etc. The development of a new generation of gene sequencing methods based on solid-state nanopores, a mature single-molecule detection technology, generated significant interest [14][15][16]. ...
The translocation of DNA molecules through nanopores has attracted wide interest for single-molecule detection. However, the multiple roles of electric fields fundamentally constrain the deceleration and motion control of DNA translocation. In this paper, we show how a single anchored DNA molecule can be manipulated for repeated capture using a transmembrane pressure gradient. Continuously and slowly changing the magnitude of the pressure provided two opposite directions for the force field inside a nanopore, and we observed an anchored DNA molecule entering the nanopore throughout the process from tentative to total entry. The use of both voltage and pressure across a nanopore provides an alternative method to capture, detect and manipulate a DNA molecule at the single-molecule level.
... Biological nanopores have also been used as sensors for organic compounds and biomolecules including peptides and proteins [55][56][57][58][59][60]. Thus, diverse applications are expected in the field of medicine, food production, cosmetics, agriculture and farming, and environmental concerns, as well as safety and security. ...
This review highlights recent development of biosensors that use the functions of membrane proteins. Membrane proteins are essential components of biological membranes and have a central role in detection of various environmental stimuli such as olfaction and gustation. A number of studies have attempted for development of biosensors using the sensing property of these membrane proteins. Their specificity to target molecules is particularly attractive as it is significantly superior to that of traditional human-made sensors. In this review, we classified the membrane protein-based biosensors into two platforms: the lipid bilayer-based platform and the cell-based platform. On lipid bilayer platforms, the membrane proteins are embedded in a lipid bilayer that bridges between the protein and a sensor device. On cell-based platforms, the membrane proteins are expressed in a cultured cell, which is then integrated in a sensor device. For both platforms we introduce the fundamental information and the recent progress in the development of the biosensors, and remark on the outlook for practical biosensing applications.
... The pathway towards nanopore protein sequencing is rich of possible intermediate achievements that can be beneficial or even ground-breaking for other parallel proteomic employment of nanofluidic devices. Among others, it is worth mentioning the singlemolecule protein identification [33,110] and detection of post-translational modifications [111,112] and Dnaprotein interaction [113]. In such a burgeoning field, a crucial role can be played by a more fundamental understanding of the electrodynamical transport phenomena under confinement. ...
Proteins perform a huge number of central functions in living organisms, thus all the new techniques allowing their precise, fast and accurate characterization at single-molecule level represent certainly a burst in proteomics with important biomedical impact. In this review, we describe the recent progresses in the developing of nanopore based devices for protein sequencing. We start by a critical analysis of the main technical requirements for nanopore protein sequencing summarizing some ideas and methodologies recently appeared in the literature. In the last sections, we focus on the physical modelling of the transport phenomena occurring in nanopore based device. The multiscale nature of the problem is discussed and, in this respect, some of the main possible
computational approaches are illustrated.
... Nanopores have emerged as powerful sensing devices for single molecules [2,3], with applications in DNA sequencing [4], protein detection [1,[5][6][7][8][9], the study of protein folding [10], SNP genotyping [11], data storage [8], and DNA computing [12]. A typical setup consists of two liquid filled reservoirs connected by a nanopore with diameters down to a few nanometres. ...
Nanopore sensing is a versatile technique for the analysis of molecules on the single-molecule level. However, extracting information from data with established algorithms usually requires time-consuming checks by an experienced researcher due to inherent variability of solid-state nanopores. Here, we develop a convolutional neural network (CNN) for the fully automated extraction of information from the time-series signals obtained by nanopore sensors. In our demonstration, we use a previously published dataset on multiplexed single-molecule protein sensing. The neural network learns to classify translocation events with greater accuracy than previously possible, while also increasing the number of analysable events by a factor of five. Our results demonstrate that deep learning can achieve significant improvements in single molecule nanopore detection with potential applications in rapid diagnostics.
Solid‐state nanopore/nanochannel biosensors have rapidly advanced due to their high sensitivity, label‐free detection, and fast response. However, detecting biomarkers directly in complex biological environments, particularly whole blood, remains challenging because of nonspecific protein adsorption and nanopore/nanochannel clogging. Here, a DNA aptamer functionalized nanochannel biosensor is developed with excellent antifouling properties, achieved by coating the nanochannel surface with agarose gel. This gel coating effectively mitigates fouling in diverse biological environments while maintaining comparable sensitivity to uncoated nanochannels for detecting prostate‐specific antigen (PSA) in buffer solutions within 20 min. The biosensor exhibits a detection limit of 1 ng mL⁻¹ for PSA in human serum, matching the performance of commercial enzyme‐linked immunosorbent assay (ELISA) kits. Importantly, it successfully differentiates whole blood samples from prostate cancer patients and healthy individuals. The superior antifouling behavior is attributed to the electrically neutral, highly hydrophilic nature, and porous structure of the agarose gel, which prevents the adsorption of large biomolecules while facilitating the diffusion of PSA for aptamer‐based capture. This DNA aptamer functionalized nanochannel biosensor with agarose gel coating offers reliable protein detection in complex biological environments, showing great promise in biomedical applications.
Protein nanopores have proven to be effective for single-molecule studies, particularly for single-stranded DNA (ssDNA) translocation. Previous experiments demonstrated their ability to distinguish differences in purine and pyrimidine bases and...
Nanopore sensing is at the forefront of the technological revolution of the protein research field and has been widely used in molecular diagnosis, molecular dynamics, as well as for various...
Biomolecular interactions, including protein‐protein, protein‐nucleic acid, and protein/nucleic acid‐ligand interactions, play crucial roles in various cellular signaling and biological processes, and offer attractive therapeutic targets in numerous human diseases. Currently, drug discovery is limited by the low efficiency and high cost of conventional ensemble‐averaging‐based techniques for biomolecular interaction analysis and high‐throughput drug screening. Nanopores are an emerging technology for single‐molecule sensing of biomolecules. Owing to the robust advantages of single‐molecule sensing, nanopore sensors have contributed tremendously to nucleic acid sequencing and disease diagnostics. In this minireview, we summarize the recent developments and outlooks in single‐molecule sensing of various biomolecular interactions for drug discovery applications using biological and solid‐state nanopore sensors.
CpG islands recruit MLL1 via the CXXC domain to modulate chromatin structure and regulate gene expression. The amino acid motif of CXXC also plays a pivotal role in MLL1's structure and function and serves as a target for drug design. In addition, the CpG pattern in an island governs spatially-dependent collaboration among CpGs in recruiting epigenetic enzymes. However, current studies using short DNA fragments cannot probe the dynamics of CXXC on long DNA with crowded CpG motifs. Here, we used single-molecule magnetic tweezers to examine the binding dynamics of MLL1's CXXC domain on a long DNA with a CpG island. The mechanical strand-separation assay allows profiling of protein-DNA complexes and reports force-dependent unfolding times. Further design of a hairpin detector reveals the unfolding time of individual CXXC-CpG complexes. Finally, in a proof of concept we demonstrate the inhibiting effect of dimethyl fumarate on the CXXC-DNA complexes by measuring the dose-response curve of the unfolding time. This demonstrates the potential feasibility of using single-molecule strand separation as a label-free detector in drug discovery and chemical biology.
The advent of single-molecule probing techniques has revolutionized the biomedical and life science fields and has spurred the development of a new class of labs-on-chip based on powerful biosensors. Nanopores represent one of the most recent and most promising single molecule sensing paradigms that is seeing increased chip-scale integration for improved convenience and performance. Due to their physical structure, nanopores are highly sensitive, require low sample volume, and offer label-free, amplification-free, high-throughput real-time detection and identification of biomolecules. Over the last 25 years, nanopores have been extensively employed to detect a variety of biomolecules with a growing range of applicatons ranging from nucleic acid sequencing to ultrasensitive diagnostics to single-molecule biophysics. Nanopores, in particular those in solid-state membranes, also have the potential for integration with other technologies such as optics, plasmonics, microfluidics, and optofluidics to perform more complex tasks for an ever-expanding demand. A number of breakthrough results using integrated nanopore platforms have already been reported, and more can be expected as nanopores remain the focus of innovative research and are finding their way into commercial instruments. This review provides an overview of different aspects and challenges of nanopore technology with a focus on chip-scale integration of solid-state nanopores for biosensing and bioanalytical applications.
The controlled dielectric breakdown emerged as a promising alternative toward accessible solid-state nanopore fabrication. Several prior studies have shown that laser-assisted dielectric breakdown could help control the nanopore position and reduce the possibility of forming multiple pores. Here, we developed a physical model to estimate the probability of forming a single nanopore under different combinations of the laser power and the electric field. This model relies on the material- and experiment-specific parameters: the Weibull statistical parameters and the laser-induced photothermal etching rate. Both the model and our experimental data suggest that a combination of a high laser power and a low electric field is statistically favorable for forming a single nanopore at a programmed location. While this model relies on experiment-specific parameters, we anticipate it could provide the experimental insights for nanopore fabrication by the laser-assisted dielectric breakdown method, enabling broader access to solid-state nanopores and their sensing applications.
Nanopore technology enables the detection and analysis of single protein molecules. The technique measures the ionic current passing through a single pore inserted in an electrically insulating membrane. The translocation of the protein molecule through the pore causes a modulation of the ionic current. Analysis of the ionic current reveals the biophysics of co-translocational unfolding and may be used to infer the amino acid sequence and posttranslational modifications of the molecule. © Springer Science+Business Media, LLC, part of Springer Nature 2021.
Nanopore analysis based on the resistive-pulse technique is an attractive tool for the single molecule detection in different fields, but it suffers the great drawback in selectivity. The common solution of this challenge is to add extra sensing aptamers and labels to analytes by improving the sensitivity of their pulses for distinguishment. Comparing to the labelling methods, we alternatively develop and demonstrate a novel data process for label-free nanopore analysis that enables the conversion of resistive current signals to more specific frequency domain phase angle features with the contribution from both sinusoidal voltage excitation and Fourier transform. In particular, we find that the transmural capacitance induced by nanoparticles translocations under a sinusoidal voltage plays an important role in this process, making phase angle features more pronounced. In practical applications, the method is successfully applied to directly distinguish the translocation events through a nanopipette by their unique phase angles for similarly sized SiO2, Ag and Au nanoparticles and soft living organisms of HeLa and LoVo, respectively, and even in a more complicated case of a SiO2, Ag and Au nanoparticle mixture.
Herein, we apply electrogenerated chemiluminescence (ECL) based method employing diaminoterephthalate analogue as ECL emitter and hairpin DNA as amplification strategy, for sensitive assay of transcription factors, which manifests enormous potential...
The increase in demand for continuous and real-time monitoring of permeation of biomolecules is addressed by using highly selective ultrathin silicon nanoporous membranes (SNMs) combined with detection using ultraviolet absorption. The membranes, with an average pore size of 8.8 nm, are fabricated using semiconductor batch processes including chemical vapor deposition and rapid thermal annealing. Bovine serum albumin (BSA) of a concentration of
/ml is used as the test molecule. The concentration of BSA diffused through the membrane is measured using optical transduction based in-house developed sensor. The photocurrent obtained from the sensor is measured every 15 min and compared with the standard Bradford assay at the same time-stamp. The concentration estimated by the sensor is found to agree with the Bradford assay with a standard deviation of 1.4%. The throughput of the membrane is increased by fabricating an array of SNMs, which showed an increase in diffusion rate by 3.8 times with respect to the single SNM. The clogging of pores by the biomolecules is analyzed using ionic conductivity experiments. The structural integrity of BSA diffused through the SNM is also analyzed.
The capture and translocation of biomolecules through nanometer-scale pores are processes with a potential large number of applications, and hence they have been intensively studied in the recent years. The aim of this paper is to review existing models of the capture process by a nanopore, together with some recent experimental data of short single- and double-stranded DNA captured by Cytolysin~A (ClyA) nanopore. ClyA is a transmembrane protein of bacterial origin which has been recently engineered through site-specific mutations, to allow the translocation of double- and single-stranded DNA. A comparison between theoretical estimations and experiments suggests that for both cases the capture is a reaction-limited process. This is corroborated by the observed salt dependence of the capture rate, which we find to be in quantitative agreement with the theoretical predictions.
Protein biomarkers in blood have been widely used in the early diagnosis of disease. However, simultaneous detection of many biomarkers in a single sample remains challenging. Here, we show that the combination of a sandwich assay and DNA‐assisted nanopore sensing could unambiguously identify several antigens in a mixture and quantify them individually. We use five barcode DNAs to label different gold nanoparticles which can selectively bind specific antigens. After the completion of the sandwich assay, barcode DNAs are released and subject to nanopore translocation tests. The distinct current signatures generated by each barcode DNA allow simultaneous quantification of biomarkers at picomolar level in clinical samples. This approach would be very useful for accurate and multiplexed quantification of cancer‐associated biomarkers within a tiny sample volume, which is critical for non‐invasive early diagnosis of cancer.
Protein biomarkers in blood have been widely used in the early diagnosis of disease. However, simultaneous detection of many biomarkers in a single sample remains challenging. Here, we show that the combination of a sandwich assay and DNA‐assisted nanopore sensing could unambiguously identify several antigens in a mixture and quantify them individually. We use five barcode DNAs to label different gold nanoparticles which can selectively bind specific antigens. After the completion of the sandwich assay, barcode DNAs are released and subject to nanopore translocation tests. The distinct current signatures generated by each barcode DNA allow simultaneous quantification of biomarkers at picomolar level in clinical samples. This approach would be very useful for accurate and multiplexed quantification of cancer‐associated biomarkers within a tiny sample volume, which is critical for non‐invasive early diagnosis of cancer.
Obtaining artificial proteins that mimic the DNA binding properties of natural transcription factors could open new ways of manipulating gene expression at will. In this context it is particularly interesting to develop simple synthetic systems. Inspired by the modularity of natural transcription factors, we have designed synthetic miniproteins that combine the zinc finger module of the transcription factor GAGA and AT-hook peptide domains. These constructs are capable of binding to composite DNA sequences of up to 14 base pairs with high affinity and good selectivity. In particular, we have synthesized three different chimeras and characterized their DNA binding properties by electrophoresis and fluorescence anisotropy. We have also used, for the first time in the study of peptide-based DNA binders, nanopore force spectroscopy to obtain further data on the DNA interaction.
Nanopore sensing is developing into a powerful single-molecule approach to investigate the features of biomolecules that are not accessible by studying ensemble systems. When a target molecule is transported through a nanopore, the ions occupying the pore are excluded, resulting in an electrical signal from the intermittent ionic blockade event. By statistical analysis of the amplitudes, duration, frequencies, and shapes of the blockade events, many properties of the target molecule can be obtained in real time at the single-molecule level, including its size, conformation, structure, charge, geometry, and interactions with other molecules. With the development of the use of α-hemolysin to characterize individual polynucleotides, nanopore technology has attracted a wide range of research interest in the fields of biology, physics, chemistry, and nanoscience. As a powerful single-molecule analytical method, nanopore technology has been applied for the detection of various biomolecules, including oligonucleotides, peptides, oligosaccharides, organic molecules, and disease-related proteins.
Here, we show a designed solid-state nanopore sensor for the direct sensing and quantification of prostate specific antigen (PSA) as cancer biomarker in serum without any pretreatment. This provides nanopore...
To achieve accurate detection of cancer biomarkers with nanopore sensors, the precise recognition of multi-level current blockage events (signature) is a pivotal problem. However, it remains rather challenging to identify the multi-level current blockages of target biomarker in nanopore experiments, especially for the nanopore analysis of serum sample. In this work, we combined a modified DBSCAN (Density-Based Spatial Clustering of Applications with Noise) algorithm with the Viterbi training algorithm to achieve intelligent retrieval of multi-level current signatures from microRNA in in serum sample. The results showed that the developed intelligent data analysis method is highly efficient for processing the large-scale nanopore data, which facilitates future application of nanopore to the clinical detection of cancer biomarkers.
A function for multisite phosphorylation
Many transcription factors are regulated by phosphorylation on multiple residues. Mylona et al. analyzed multisite phosphorylation in the transcription factor Elk-1 and showed that it may protect against excessive activation (see the Perspective by Whitmarsh and Davis). Phosphorylation by the kinase ERK2 occurred at eight sites, but the sites were phosphorylated at different rates. Those that were phosphorylated more quickly promoted transcriptional activation. Those that were phosphorylated more slowly dampened excessive activation by ERK2s without needing a phosphatase or any other negative regulatory component.
Science , this issue p. 233 ; see also p. 179
Protein engineering has been used to remodel pores for applications in biotechnology. For example, the heptameric α-hemolysin pore (αHL) has been engineered to form a nanoreactor to study covalent chemistry at the single-molecule level. Previous work has been confined largely to the chemistry of cysteine side-chains, or in one instance to an irreversible reaction of an unnatural amino acid side-chain bearing a terminal alkyne. Here, we present four different αHL pores obtained by coupling either two or three fragments by native chemical ligation (NCL). The synthetic αHL monomers were folded and incorporated into heptameric pores. The functionality of the pores was validated by hemolysis assays and by single-channel current recording. By using NCL to introduce a ketone amino acid, the nanoreactor approach was extended to an investigation of reversible covalent chemistry on an unnatural side-chain at the single-molecule level.
Gene expression is regulated by transcription factors (TFs), proteins that recognize short DNA sequence motifs. Such sequences are very common in the human genome, and an important determinant of the specificity of gene expression is the cooperative binding of multiple TFs to closely located motifs. However, interactions between DNA-bound TFs have not been systematically characterized. To identify TF pairs that bind cooperatively to DNA, and to characterize their spacing and orientation preferences, we have performed consecutive affinity-purification systematic evolution of ligands by exponential enrichment (CAP-SELEX) analysis of 9,400 TF-TF-DNA interactions. This analysis revealed 315 TF-TF interactions recognizing 618 heterodimeric motifs, most of which have not been previously described. The observed cooperativity occurred promiscuously between TFs from diverse structural families. Structural analysis of the TF pairs, including a novel crystal structure of MEIS1 and DLX3 bound to their identified recognition site, revealed that the interactions between the TFs were predominantly mediated by DNA. Most TF pair sites identified involved a large overlap between individual TF recognition motifs, and resulted in recognition of composite sites that were markedly different from the individual TF's motifs. Together, our results indicate that the DNA molecule commonly plays an active role in cooperative interactions that define the gene regulatory lexicon.
Transcription factor shape-shifts DNA
Controlling when a gene is expressed or repressed is vital for cellular metabolism and development. In Escherichia coli , a copper-sensing transcription factor, CueR, can repress or activate expression when bound at exactly the same site in the gene promoter. Philips et al. used x-ray crystallography to show that CueR dramatically changes the topology of the DNA upon binding copper but does not affect protein-DNA contacts. The DNA shape change allows RNA polymerase to bind the promoter and transcribe the gene.
Science , this issue p. 877
Transcription factor (TF)-DNA interactions are the primary control point in regulation of gene expression. Characterization of these interactions is essential for understanding genetic regulation of biological systems and developing novel therapies to treat cellular malfunctions. Solid-state nanopores are a highly versatile class of single-molecule sensors that can provide rich information about local properties of long charged biopolymers using the current blockage patterns generated during analyte translocation, and provide a novel platform for characterization of TF-DNA interactions. The DNA-binding domain of the TF Early Growth Response Protein 1 (EGR1), a prototypical zinc finger protein known as zif268, is used as a model system for this study. zif268 adopts two distinct bound conformations corresponding to specific and nonspecific binding, according to the local DNA sequence. Here we implement a solid-state nanopore platform for direct, label- and tether-free single-molecule detection of zif268 bound to DNA. We demonstrate detection of single zif268 TFs bound to DNA according to current blockage sublevels and duration of translocation through the nanopore. We further show that the nanopore can detect and discriminate both specific and nonspecific binding conformations of zif268 on DNA via the distinct current blockage patterns corresponding to each of these two known binding modes.
Understanding how eukaryotic enhancers are bound and regulated by specific combinations of transcription factors is still a major challenge. To better map transcription factor binding genome-wide at nucleotide resolution in vivo, we have developed a robust ChIP-exo protocol called ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode and single ligation), which utilizes an efficient DNA self-circularization step during library preparation. Application of ChIP-nexus to four proteins-human TBP and Drosophila NFkB, Twist and Max-shows that it outperforms existing ChIP protocols in resolution and specificity, pinpoints relevant binding sites within enhancers containing multiple binding motifs, and allows for the analysis of in vivo binding specificities. Notably, we show that Max frequently interacts with DNA sequences next to its motif, and that this binding pattern correlates with local DNA-sequence features such as DNA shape. ChIP-nexus will be broadly applicable to the study of in vivo transcription factor binding specificity and its relationship to cis-regulatory changes in humans and model organisms.
In nanopore sequencing, where single DNA strands are electrophoretically translocated through a nanopore and the resulting ionic signal is used to identify the four DNA bases, an enzyme has been used to ratchet the nucleic acid stepwise through the pore at a controlled speed. In this work, we investigated the ability of alpha-hemolysin nanopores to distinguish the four DNA bases under conditions that are compatible with the activity of DNA-handling enzymes. Our findings suggest that in immobilized strands, the applied potential exerts a force on DNA (∼10 pN at +160 mV) that increases the distance between nucleobases by about 2.2 Å V(-1). The four nucleobases can be resolved over wide ranges of applied potentials (from +60 to +220 mV in 1 m KCl) and ionic strengths (from 200 mM KCl to 1 M KCl at +160 mV) and nucleobase recognition can be improved when the ionic strength on the side of the DNA-handling enzyme is low, while the ionic strength on the opposite side is high.
Nanopores are a versatile technique for the detection and characterisation of single molecules in solution. An ongoing challenge in the field is to find methods to selectively detect specific biomolecules. In this work we describe a new technique for sensing specific proteins using unmodified solid-state nanopores. We engineered a double strand of DNA by hybridising nearly two hundred oligonucleotides to a linearised version of the m13mp18 virus genome. This engineered double strand, which we call a DNA carrier, allows positioning of protein binding sites at nanometre accurate intervals along its contour via DNA conjugation chemistry. We measure the ionic current signal of translocating DNA carriers as a function of the number of binding sites and show detection down to the single protein level. Furthermore, we use DNA carriers to develop an assay for identifying a single protein species within a protein mixture.
The transcription factor FOXM1 binds to sequence-specific motifs on DNA (C/TAAACA) through its DNA-binding domain (DBD) and activates proliferation- and differentiation-associated genes. Aberrant overexpression of FOXM1 is a key feature in oncogenesis and progression of many human cancers. Here-from a high-throughput screen applied to a library of 54,211 small molecules-we identify novel small molecule inhibitors of FOXM1 that block DNA binding. One of the identified compounds, FDI-6 (NCGC00099374), is characterized in depth and is shown to bind directly to FOXM1 protein, to displace FOXM1 from genomic targets in MCF-7 breast cancer cells, and induce concomitant transcriptional downregulation. Global transcript profiling of MCF-7 cells by RNA-seq shows that FDI-6 specifically downregulates FOXM1-activated genes with FOXM1 occupancy confirmed by ChIP-PCR. This small molecule-mediated effect is selective for FOXM1-controlled genes with no effect on genes regulated by homologous forkhead family factors.
Protein unfolding and translocation through pores occurs during trafficking between organelles, protein degradation and bacterial toxin delivery. In vivo, co-translocational unfolding can be affected by the end of the polypeptide that is threaded into the pore first. Recently, we have shown that co-translocational unfolding can be followed in a model system at the single-molecule level, thereby unravelling molecular steps and their kinetics. Here, we show that the unfolding kinetics of the model substrate thioredoxin, when pulled through an α-haemolysin pore, differ markedly depending on whether the process is initiated from the C terminus or the N terminus. Further, when thioredoxin is pulled from the N terminus, the unfolding pathway bifurcates: some molecules finish unfolding quickly, while others finish ~100 times slower. Our findings have important implications for the understanding of biological unfolding mechanisms and in the application of nanopore technology for the detection of proteins and their modifications.
Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme-DNA complex by depleting Mg(2+)-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity, but in fact reflects one microstate from the equilibrium ensemble that a single molecule manifests during an individual processive translocation event.
Cells are divided into compartments and separated from the environment by lipid bilayer membranes. Essential molecules are transported back and forth across the membranes. We have investigated how folded proteins use narrow transmembrane pores to move between compartments. During this process, the proteins must unfold. To examine co-translocational unfolding of individual molecules, we tagged protein substrates with oligonucleotides to enable potential-driven unidirectional movement through a model protein nanopore, a process that differs fundamentally from extension during force spectroscopy measurements. Our findings support a four-step translocation mechanism for model thioredoxin substrates. First, the DNA tag is captured by the pore. Second, the oligonucleotide is pulled through the pore, causing local unfolding of the C terminus of the thioredoxin adjacent to the pore entrance. Third, the remainder of the protein unfolds spontaneously. Finally, the unfolded polypeptide diffuses through the pore into the recipient compartment. The unfolding pathway elucidated here differs from those revealed by denaturation experiments in solution, for which two-state mechanisms have been proposed.
Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules. Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates. In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.
Several methods for characterizing DNA-protein interactions are available, but none have demonstrated both high throughput and quantitative measurement of affinity. Here we describe 'high-throughput sequencing'-'fluorescent ligand interaction profiling' (HiTS-FLIP), a technique for measuring quantitative protein-DNA binding affinity at unprecedented depth. In this approach, the optics built into a high-throughput sequencer are used to visualize in vitro binding of a protein to sequenced DNA in a flow cell. Application of HiTS-FLIP to the protein Gcn4 (Gcn4p), the master regulator of the yeast amino acid starvation response, yielded ~440 million binding measurements, enabling determination of dissociation constants for all 12-mer sequences having submicromolar affinity. These data revealed a complex interdependency between motif positions, allowed improved discrimination of in vivo Gcn4p binding sites and regulatory targets relative to previous methods and showed that sets of genes with different promoter affinities to Gcn4p have distinct functions and expression kinetics. Broad application of this approach should increase understanding of the interactions that drive transcription.
FoxM1 is a member of the Forkhead family of transcription factors and is implicated in inducing cell proliferation and some
forms of tumorigenesis. It binds promoter regions with a preference for tandem repeats of a consensus ‘TAAACA’ recognition
sequence. The affinity of the isolated FoxM1 DNA-binding domain for this site is in the micromolar range, lower than observed
for other Forkhead proteins. To explain these FoxM1 features, we determined the crystal structure of its DNA-binding domain
in complex with a tandem recognition sequence. FoxM1 adopts the winged-helix fold, typical of the Forkhead family. Neither
‘wing’ of the fold however, makes significant contacts with the DNA, while the second, C-terminal, wing adopts an unusual
ordered conformation across the back of the molecule. The lack of standard DNA–‘wing’ interactions may be a reason for FoxM1’s
relatively low affinity. The role of the ‘wings’ is possibly undertaken by other FoxM1 regions outside the DBD, that could
interact with the target DNA directly or mediate interactions with other binding partners. Finally, we were unable to show
a clear preference for tandem consensus site recognition in DNA-binding, transcription activation or bioinformatics analysis;
FoxM1's moniker, ‘Trident’, is not supported by our data.
The sequencing of individual DNA strands with nanopores is under investigation as a rapid, low-cost platform in which bases are identified in order as the DNA strand is transported through a pore under an electrical potential. Although the preparation of solid-state nanopores is improving, biological nanopores, such as alpha-hemolysin (alphaHL), are advantageous because they can be precisely manipulated by genetic modification. Here, we show that the transmembrane beta-barrel of an engineered alphaHL pore contains 3 recognition sites that can be used to identify all 4 DNA bases in an immobilized single-stranded DNA molecule, whether they are located in an otherwise homopolymeric DNA strand or in a heteropolymeric strand. The additional steps required to enable nanopore DNA sequencing are outlined.
The alpha-hemolysin gene from Staphylococcus aureus, excluding the 5' region encoding the hydrophobic leader sequence, was amplified from genomic DNA. The identity of the disputed C terminus has been confirmed and revisions made to the internal sequence. The hemolysin is expressed at high levels in Escherichia coli and has been purified to homogeneity from this source. In addition, active [35S-Met]alpha-hemolysin of high specific radioactivity can be generated in an E. coli transcription-translation system. By criteria based on protein chemistry, and biological and electrophysiological assays, the recombinant polypeptide is closely similar to the staphylococcal polypeptide ruling out the possibility of functionally important posttranslational modifications in S. aureus. Convenient new assays utilizing the 35S-labeled polypeptide to measure erythrocyte binding, oligomer formation in detergent and on target cells, and hemolysis have been developed. They have been used to demonstrate that a deletion mutant of alpha-hemolysin, in which five C-terminal amino acids are absent, is severely compromised in its ability both to oligomerize and to lyse rabbit erythrocytes. The mutant polypeptide nevertheless binds tightly to erythrocytes as a monomer, strengthening the idea that oligomerization is required for cell lysis.
Intercalating, minor groove binding, and covalently bonding drugs were evaluated by mobility shift assays for their ability to interfere with transcription factors binding to their respective DNA recognition sequences. The Cys2His2 zinc finger proteins EGR1, WT1, and NIL2A, the basic leucine-zipper protein wbJun/wbFos, and the minor groove binding protein hTBP were chosen as representative transcription factors. Their DNA recognition sites include G/C-rich, mixed, and A/T-rich sequences. The intercalators nogalamycin and hedamycin, and the G/C-specific minor groove binding drug chromomycin A3 were the most potent drugs, preventing transcription factor.DNA complex formation at concentrations less than 1 microM. Similar concentrations of chromomycin A3 disrupted preformed complexes while nogalamycin and hedamycin were 50-fold less potent if proteins were allowed to bind DNA prior to drug treatment. Echinomycin inhibited EGR1.DNA complex formation 50% at 5 microM but had little effect on the formation of NIL2A.DNA complexes. Conversely, doxorubicin was found to inhibit NIL2A complex formation 50% at less than 1 microM, but did not achieve this level of inhibition of EGR1/DNA complex formation even at 50 microM. The A/T-directed minor groove binding drugs, while inhibiting hTBP at submicromolar concentrations, had no effect on either EGR1 or NIL2A.
The initiator protein (RepE) of F factor, a plasmid involved in sexual conjugation in Escherichia coli, has dual functions during the initiation of DNA replication which are determined by whether it exists as a dimer or as a monomer. A RepE monomer functions as a replication initiator, but a RepE dimer functions as an autogenous repressor. We have solved the crystal structure of the RepE monomer bound to an iteron DNA sequence of the replication origin of plasmid F. The RepE monomer consists of topologically similar N- and C-terminal domains related to each other by internal pseudo 2-fold symmetry, despite the lack of amino acid similarities between the domains. Both domains bind to the two major grooves of the iteron (19 bp) with different binding affinities. The C-terminal domain plays the leading role in this binding, while the N-terminal domain has an additional role in RepE dimerization. The structure also suggests that superhelical DNA induced at the origin of plasmid F by four RepEs and one HU dimer has an essential role in the initiation of DNA replication.
A variety of different DNA polymers were electrophoretically driven through the nanopore of an alpha-hemolysin channel in a lipid bilayer. Single-channel recording of the translocation duration and current flow during traversal of individual polynucleotides yielded a unique pattern of events for each of the several polymers tested. Statistical data derived from this pattern of events demonstrate that in several cases a nanopore can distinguish between polynucleotides of similar length and composition that differ only in sequence. Studies of temperature effects on the translocation process show that translocation duration scales as approximately T(-2). A strong correlation exists between the temperature dependence of the event characteristics and the tendency of some polymers to form secondary structure. Because nanopores can rapidly discriminate and characterize unlabeled DNA molecules at low copy number, refinements of the experimental approach demonstrated here could eventually provide a low-cost high-throughput method of analyzing DNA polynucleotides.
Nanoscale α‐hemolysin pores can be used to analyze individual DNA or RNA molecules. Serial examination of hundreds to thousands
of molecules per minute is possible using ionic current impedance as the measured property. In a recent report, we showed
that a nanopore device coupled with machine learning algorithms could automatically discriminate among the four combinations
of Watson–Crick base pairs and their orientations at the ends of individual DNA hairpin molecules. Here we use kinetic analysis
to demonstrate that ionic current signatures caused by these hairpin molecules depend on the number of hydrogen bonds within
the terminal base pair, stacking between the terminal base pair and its nearest neighbor, and 5′ versus 3′ orientation of
the terminal bases independent of their nearest neighbors. This report constitutes evidence that single Watson–Crick base
pairs can be identified within individual unmodified DNA hairpin molecules based on their dynamic behavior in a nanoscale
pore.
Members of the DExH/D superfamily of nucleic acid–activated nucleotide triphosphatases are essential for virtually all aspects
of RNA metabolism, including pre–messenger RNA splicing, RNA interference, translation, and nucleocytoplasmic trafficking.
Physiological substrates for these enzymes are thought to be regions of double-stranded RNA, because several DExH/D proteins
catalyze strand separation in vitro. These “RNA helicases” can also disrupt RNA-protein interactions, but it is unclear whether
this activity is coupled to duplex unwinding. Here we demonstrate that two unrelated DExH/D proteins catalyze protein displacement
independently of duplex unwinding. Therefore, the essential functions of DExH/D proteins are not confined to RNA duplexes
but can be exerted on a wide range of ribonucleoprotein substrates.
The simultaneous detection of a large number of different analytes is important in bionanotechnology research and in diagnostic applications. Nanopore sensing is an attractive method in this regard as the approach can be integrated into small, portable device architectures, and there is significant potential for detecting multiple sub-populations in a sample. Here, we show that highly multiplexed sensing of single molecules can be achieved with solid-state nanopores by using digitally encoded DNA nanostructures. Based on the principles of DNA origami, we designed a library of DNA nanostructures in which each member contains a unique barcode; each bit in the barcode is signalled by the presence or absence of multiple DNA dumbbell hairpins. We show that a 3-bit barcode can be assigned with 94% accuracy by electrophoretically driving the DNA structures through a solid-state nanopore. Select members of the library were then functionalized to detect a single, specific antibody through antigen presentation at designed positions on the DNA. This allows us to simultaneously detect four different antibodies of the same isotype at nanomolar concentration levels.
Cancer, more than any other human disease, now has a surfeit of potential molecular targets poised for therapeutic exploitation. Currently, a number of attractive and validated cancer targets remain outside of the reach of pharmacological regulation. Some have been described as undruggable, at least by traditional strategies. In this article, we outline the basis for the undruggable moniker, propose a reclassification of these targets as undrugged, and highlight three general classes of this imposing group as exemplars with some attendant strategies currently being explored to reclassify them. Expanding the spectrum of disease-relevant targets to pharmacological manipulation is central to reducing cancer morbidity and mortality. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 56 is January 06, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Portable device offers on-the-spot data to fight disease, catalogue species and more.
Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets.
Copyright © 2015 Elsevier Inc. All rights reserved.
Protein-DNA interactions play critical roles in biological systems, and they often involve complex mechanisms and dynamics that are not easily measured by ensemble experiments. Recently, we showed that folded proteins can be internalised inside ClyA nanopores and studied by ionic current recordings at the single-molecule level. Here, we use ClyA nanopores to sample the interaction between the G-quadruplex fold of the thrombin binding aptamer (TBA) and human thrombin (HT). Surprisingly, the internalisation of the HT:TBA complex inside the nanopore induced two types of current blockades with distinguished residual current and lifetime. Using single nucleobase substitutions to TBA we showed that these two types of blockades originate from TBA binding to thrombin with two isomeric orientations. Voltage dependencies and the use of ClyA nanopores with two different diameters allowed assessing the effect of the applied potential and confinement, and revealed that the two binding configurations of TBA to HT display different lifetimes. These results show that the ClyA nanopores can be used to probe conformational heterogeneity in protein:DNA interactions.
Analysis of the origins of new drugs approved by the US Food and Drug Administration (FDA) from 1999 to 2008 suggested that phenotypic screening strategies had been more productive than target-based approaches in the discovery of first-in-class small-molecule drugs. However, given the relatively recent introduction of target-based approaches in the context of the long time frames of drug development, their full impact might not yet have become apparent. Here, we present an analysis of the origins of all 113 first-in-class drugs approved by the FDA from 1999 to 2013, which shows that the majority (78) were discovered through target-based approaches (45 small-molecule drugs and 33 biologics). In addition, of 33 drugs identified in the absence of a target hypothesis, 25 were found through a chemocentric approach in which compounds with known pharmacology served as the starting point, with only eight coming from what we define here as phenotypic screening: testing a large number of compounds in a target-agnostic assay that monitors phenotypic changes. We also discuss the implications for drug discovery strategies, including viewing phenotypic screening as a novel discipline rather than as a neoclassical approach.
Herein we report a novel approach for fast, label-free probing of DNA-histone interactions in individual nucleosomes. We use solid-state nanopores to unravel individual DNA/histone complexes for the first time, and find that the unraveling time depends on the applied electrophoretic force, and our results are in line with previous studies that employ optical tweezers. Our approach for studying nucleosomal interactions can greatly accelerate our understanding of fundamental mechanisms by which transcription, replication, and repair processes in a cell are modulated through DNA-histone interactions, as well as in diagnosis of diseases with abnormal patterns of DNA and histone modifications.
In this protocol, we describe a procedure to generate 'DNA dumbbells'-single molecules of DNA with a microscopic bead attached at each end-and techniques for manipulating individual DNA dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA dumbbells and to visualize individual protein-DNA complexes by single-molecule epifluorescence microscopy. Our design of the flow cell enables the rapid movement of trapped molecules between laminar flow channels and a flow-free reservoir. The reservoir provides the means to examine the formation of protein-DNA complexes in solution in the absence of external flow forces while maintaining a predetermined end-to-end extension of the DNA. These features facilitate the examination of the role of 3D DNA conformation and dynamics in protein-DNA interactions. Preparation of flow cells and reagents requires 2 days each; in situ DNA dumbbell assembly and imaging of single protein-DNA complexes require another day.
Using nanopores to sequence biopolymers was proposed more than a decade ago. Recent advances in enzyme-based control of DNA translocation and in DNA nucleotide resolution using modified biological pores have satisfied two technical requirements of a functional nanopore DNA sequencing device. Nanopore sequencing of proteins was also envisioned. Although proteins have been shown to move through nanopores, a technique to unfold proteins for processive translocation has yet to be demonstrated. Here we describe controlled unfolding and translocation of proteins through the α-hemolysin (α-HL) pore using the AAA+ unfoldase ClpX. Sequence-dependent features of individual engineered proteins were detected during translocation. These results demonstrate that molecular motors can reproducibly drive proteins through a model nanopore-a feature required for protein sequence analysis using this single-molecule technology.
AAA(+) unfoldases denature and translocate polypeptides into associated peptidases. We report direct observations of mechanical, force-induced protein unfolding by the ClpX unfoldase from E. coli, alone, and in complex with the ClpP peptidase. ClpX hydrolyzes ATP to generate mechanical force and translocate polypeptides through its central pore. Threading is interrupted by pauses that are found to be off the main translocation pathway. ClpX's translocation velocity is force dependent, reaching a maximum of 80 aa/s near-zero force and vanishing at around 20 pN. ClpX takes 1, 2, or 3 nm steps, suggesting a fundamental step-size of 1 nm and a certain degree of intersubunit coordination. When ClpX encounters a folded protein, it either overcomes this mechanical barrier or slips on the polypeptide before making another unfolding attempt. Binding of ClpP decreases the slip probability and enhances the unfolding efficiency of ClpX. Under the action of ClpXP, GFP unravels cooperatively via a transient intermediate.
Nanopore force spectroscopy is used to study the unzipping kinetics of two DNA hairpin molecules with a 12 base pair long stem containing two contiguous stretches of six GC and six AT base pairs in interchanged order. Even though the thermodynamic stabilities of the two structures are nearly the same, they differ greatly in their unzipping kinetics. When the GC segment has to be broken before the AT segment, the unfolding rate is orders of magnitude smaller than in the opposite case. We also investigated hairpins with stem regions consisting only of AT or GC base pairs. The pure AT hairpins translocate much faster than the other hairpins, whereas the pure GC hairpins translocate on similar timescales to the hairpins with only an initial GC segment. For each hairpin, nanopore force spectroscopy is performed for different loading rates and the resulting unzipping distributions are mathematically transformed to a master curve that yields the unfolding rate as a function of applied voltage. This is compared with a stochastic model of the unfolding process for the two sequences for different voltages. The results can be rationalized in terms of the different natures of the free energy landscapes for the unfolding process.
Single-molecule fluorescence techniques have emerged as powerful tools to study biological processes at the molecular level. This review describes the application of these methods to the characterization of the kinetics of interaction between biomolecules. A large number of single-molecule assays have been developed that visualize association and dissociation kinetics in vitro by fluorescently labeling binding partners and observing their interactions over time. Even though recent progress has been significant, there are certain limitations to this approach. To allow the observation of individual, fluorescently labeled molecules requires low, nanomolar concentrations. I will discuss how such concentration requirements in single-molecule experiments limit their applicability to investigate intermolecular interactions and how recent technical advances deal with this issue.
We describe the methods used in our laboratory for the analysis of single nucleic acid molecules with protein nanopores. The technical section is preceded by a review of the variety of experiments that can be done with protein nanopores. The end goal of much of this work is single-molecule DNA sequencing, although sequencing is not discussed explicitly here. The technical section covers the equipment required for nucleic acid analysis, the preparation and storage of the necessary materials, and aspects of signal processing and data analysis.
We present a novel single-molecule method for rapidly evaluating small-molecule binding to individual DNA molecules using nanopores fabricated in ultrathin silicon membranes. A measurable shift in the residual ion current through a approximately 3.5 nm pore results from threading of a dye-intercalated DNA molecule, as compared to the typical residual current of native DNA. The average level of the residual current can be used to directly quantify the fraction of bound molecules to DNA, providing a new way to determine binding isotherms. Spatial sensitivity is also demonstrated by designing a two-segment DNA molecule that contains small-molecule binding sites in one of its two segments. Translocations of such molecules exhibit two current levels upon incubation with a DNA-binding dye, caused by selectively bound dye in one of the DNA segments. Our results, as shown here with four different dyes, coincide well with bulk fluorescence measurements performed under identical conditions. The nanopore approach for "reading-out" molecular binding along a DNA molecule, combined with the miniscule amounts of DNA required and the potential for scalability using nanopore arrays, provide a novel platform for future applications in analytical drug screening.
We report translocation of double-stranded DNA (dsDNA) molecules that are coated with RecA protein through solid-state nanopores. Translocation measurements show current-blockade events with a wide variety in time duration (10-4-10-1 s) and conductance blockade values (3-14 nS). Large blockades (11.4+/-0.7 nS) are identified as being caused by translocations of RecA-dsDNA filaments. We confirm these results through a variety of methods, including changing molecular length and using an optical tweezer system to deliver bead-functionalized molecules to the nanopore. We further distinguish two different regimes of translocation: a low-voltage regime (<150 mV) in which the event rate increases exponentially with voltage, and a high-voltage regime in which it remains constant. Our results open possibilities for a variety of future experiments with (partly) protein-coated DNA molecules, which is interesting for both fundamental science and genomic screening applications.
Replication of mini-F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherichia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dimeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization of RepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication.
It was recently shown that naturally occurring, genetically engineered or chemically modified channels can be used to detect analytes in solution. We demonstrate here that the overall range of analytes that can be detected by single nanometer-scale pores is expanded using a potentially simpler system. Instead of attaching recognition elements to a channel, they are covalently linked to polymers that otherwise thread through a nanometer-scale pore. Because the rate of unbound polymer entering the pore is proportional to its concentration in the bulk, the binding of analyte to the polymer alters the latter's ability to thread through the pore, and the signal that results from individual polymer translocation is unique to the polymer type; the method permits multianalyte detection and quantitation. We demonstrate here that two different proteins can be simultaneously detected with this technique.
We present unzipping force analysis of protein association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex. The capacity of UFAPA to spatially locate protein-DNA interactions is demonstrated by noncatalytic restriction mapping on a 4-kb DNA with three restriction enzymes (BsoBI, XhoI, and EcoRI). A restriction map for a given restriction enzyme was generated with an accuracy of approximately 25 bp. UFAPA also allows direct determination of the site-specific equilibrium association constant (K(A)) for a DNA-binding protein. This capability is demonstrated by measuring the cation concentration dependence of K(A) for EcoRI binding. The measured values are in good agreement with previous measurements of K(A) over an intermediate range of cation concentration. These results demonstrate the potential utility of UFAPA for future studies of site-specific protein-DNA interactions.
The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
We have used the nanometer scale alpha-Hemolysin pore to study the unzipping kinetics of individual DNA hairpins under constant force or constant loading rate. Using a dynamic voltage control method, the entry rate of polynucleotides into the pore and the voltage pattern applied to induce hairpin unzipping are independently set. Thus, hundreds of unzipping events can be tested in a short period of time (few minutes), independently of the unzipping voltage amplitude. Because our method does not entail the physical coupling of the molecules under test to a force transducer, very high throughput can be achieved. We used our method to study DNA unzipping kinetics at small forces, which have not been accessed before. We find that in this regime the static unzipping times decrease exponentially with voltage with a characteristic slope that is independent of the duplex region sequence, and that the intercept depends strongly on the duplex region energy. We also present the first nanopore dynamic force measurements (time varying force). Our results are in agreement with the approximately logV dependence at high V (where V is the loading rate) observed by other methods. The extension of these measurements to lower loading rates reveals a much weaker dependence on V.
We present a method for rapid measurement of DNA-protein interactions using voltage-driven threading of single DNA molecules through a protein nanopore. Electrical force applied to individual ssDNA-exonuclease I complexes pulls the two molecules apart, while ion current probes the dissociation rate of the complex. Nanopore force spectroscopy (NFS) reveals energy barriers affecting complex dissociation. This method can be applied to other nucleic acid-protein complexes, using protein or solid-state nanopore devices.
Helicases and translocases are a ubiquitous, highly diverse group of proteins that perform an extraordinary variety of functions in cells. Consequently, this review sets out to define a nomenclature for these enzymes based on current knowledge of sequence, structure, and mechanism. Using previous definitions of helicase families as a basis, we delineate six superfamilies of enzymes, with examples of crystal structures where available, and discuss these structures in the context of biochemical data to outline our present understanding of helicase and translocase activity. As a result, each superfamily is subdivided, where appropriate, on the basis of mechanistic understanding, which we hope will provide a framework for classification of new superfamily members as they are discovered and characterized.
DNA replication initiator protein RepE stringently regulates F plasmid replication by its two distinct molecular association states. A predominant dimer functions as an autogenous repressor, whereas monomers act as replication initiators, and the dimer requires actions of the DnaK molecular chaperone system for monomerization. The structure of the monomeric form is known, whereas the dimeric structure and structural details of the dimer-to-monomer conversion have been unclear. Here we present the crystal structure of the RepE dimer in complex with the repE operator DNA. The dimerization interface is mainly formed by intermolecular β-sheets with several key interactions of charged residues. The conformations of the internal N- and C-terminal domains are conserved between the dimer and monomer, whereas the relative domain orientations are strikingly different, allowing for an efficient oligomeric transition of dual-functional RepE. This domain relocation accompanies secondary structural changes in the linker connecting the two domains, and the linker is included in plausible DnaK/DnaJ-binding regions. These findings suggest an activation mechanism for F plasmid replication by RepE monomerization, which is induced and mediated by the DnaK system.
• DnaK
• dual function
• F plasmid
• RepE
• repressor–operator complex
Increasing the throughput and resolution of electrical recording of currents through ion conducting channels and pores is an important technical challenge both for the functional analysis of ion channel proteins and for the application of nanoscale pores in single molecule analytical tasks. We present a novel design based on sub-picoliter-cavities arrayed in a polymer substrate and endowed with individual planar microelectrodes that allows low-noise and parallel electrical recording from ion channels and pores. Resolution of voltage-dependent current transitions of alamethicin channels as well as polyethylene-glycol-induced blocking events of alpha-hemolysin nanopores on the submillisecond time scale is demonstrated using this device.